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Triterpenes are among the most diverse plant natural products, and their diversity is closely related to various triterpene skeletons catalyzed by different 2,3-oxidosqualene cyclases (OSCs). Celastrol, a friedelane-type triterpene with significant bioactivities, is specifically distributed in higher plants, such as Celastraceae species. Friedelin is an important precursor for the biosynthesis of celastrol, and it is synthesized through the cyclization of 2,3-oxidosqualene, with the highest number of rearrangements being catalyzed by friedelane-type triterpene cyclases. However, the molecular mechanisms underlying the catalysis of friedelin production by friedelane-type triterpene cyclases have not yet been fully elucidated. In this study, transcriptome data of four celastrol-producing plants from Celastraceae were used to identify a total of 21 putative OSCs. Through functional characterization, the friedelane-type triterpene cyclases were separately verified in the four plants. Analysis of the selection pressure showed that purifying selection acted on these OSCs, and the friedelane-type triterpene cyclases may undergo weaker selective restriction during evolution. Molecular docking and site-directed mutagenesis revealed that changes in some amino acids that are unique to friedelane-type triterpene cyclases may lead to variations in catalytic specificity or efficiency, thereby affecting the synthesis of friedelin. Our research explored the functional diversity of triterpene synthases from a multispecies perspective. It also provides some references for further research on the relative mechanisms of friedelin biosynthesis.

Summary Histone variants contribute to the complexity of the chromatin landscape and play an integral role in defining DNA domains and regulating gene expression. The histone H3 variant H3.3 is incorporated into genic elements independent of DNA replication by its chaperone HIRA. Here we demonstrate that Hira is required for the self-renewal of adult hematopoietic stem cells (HSCs) and to restrain erythroid differentiation. Deletion of Hira led to rapid depletion of HSCs while differentiated hematopoietic cells remained largely unaffected. Depletion of HSCs after Hira deletion was accompanied by increased expression of bivalent and erythroid genes, which was exacerbated upon cell division and paralleled increased erythroid differentiation. Assessing H3.3 occupancy identified a subset of polycomb-repressed chromatin in HSCs that depends on HIRA to maintain the inaccessible, H3.3-occupied state for gene repression. HIRA-dependent H3.3 incorporation thus defines distinct repressive chromatin that represses erythroid differentiation of HSCs., Graphical abstract, Highlights • Deletion of Hira depletes adult HSCs but not fetal liver HSCs • HIRA represses hematopoietic development and erythropoiesis genes in HSCs • Cell division exacerbates gene derepression in Hira-deficient HSCs • H3.3 deposition and the closed state of polycomb-repressed chromatin depend on HIRA, Murdaugh et al. demonstrate that deletion of the histone H3.3 chaperone Hira depletes adult but not fetal liver HSCs by inducing differentiation. Hira regulates H3.3 deposition at a subset of polycomb-repressed chromatin that encodes genes involved in hematopoietic development, which becomes accessible and derepressed in the absence of Hira.

Stem cells need to be protected from genotoxic and proteotoxic stress to maintain a healthy pool throughout life1–3. Little is known about the proteostasis mechanism that safeguards stem cells. Here we report endoplasmic reticulum-associated degradation (ERAD) as a protein quality checkpoint that controls the haematopoietic stem cell (HSC)–niche interaction and determines the fate of HSCs. The SEL1L–HRD1 complex, the most conserved branch of ERAD4, is highly expressed in HSCs. Deletion of Sel1l led to niche displacement of HSCs and a complete loss of HSC identity, and allowed highly efficient donor-HSC engraftment without irradiation. Mechanistic studies identified MPL, the master regulator of HSC identity5, as a bona fide ERAD substrate that became aggregated in the endoplasmic reticulum following ERAD deficiency. Restoration of MPL signalling with an agonist partially rescued the number and reconstitution capacity of Sel1l-deficient HSCs. Our study defines ERAD as an essential proteostasis mechanism to safeguard a healthy stem cell pool by regulating the stem cell–niche interaction. Xu, Liu, Pen, Zhang et al. demonstrate that the SEL1L–HRD1 complex, which is part of the ERAD protein quality control machinery, safeguards haematopoietic stem cell identity and niche location by ensuring the haematopoietic stem cell surface expression of MPL.

Summary Bone marrow stromal cells (BMSCs) that express high levels of stem cell factor (SCF) and CXC chemokine ligand 12 (CXCL12) are one crucial component of the hematopoietic stem cell (HSC) niche. While the secreted factors produced by BMSCs to support HSCs have been well described, little is known regarding the transcriptional regulators controlling the cell fate of BMSCs and thus indirectly maintaining HSCs. BMI1 is a polycomb group protein that regulates HSCs both cell intrinsically and extrinsically, but it is unknown in which cell type and how BMI1 functions to maintain HSCs extrinsically. Here we show that Bmi1 maintains HSCs by preventing adipogenic differentiation of BMSCs. Bmi1 is highly expressed in BMSCs but becomes downregulated upon adipogenic differentiation and during aging. Deleting Bmi1 from BMSCs increased marrow adipocytes, induced HSC quiescence and depletion, and impaired hematopoiesis. We found that BMI1 repressed multiple developmental programs in BMSCs by safeguarding the repressive epigenetic marks histone H2A ubiquitylation and H3 lysine 27 trimethylation. We identified a novel adipogenic program governed by Pax3, which BMI1 repressed in BMSCs. Our results establish Bmi1 as a critical regulator of BMSC cell fate that suppresses marrow adipogenesis to create a supportive niche for HSCs., Graphical Abstract, Highlights • Bmi1 prevents adipogenesis and promotes osteogenesis by bone marrow stromal cells • Adipocyte expansion after Bmi1 loss in stroma depletes hematopoietic stem cells • Loss of Bmi1 reduces repressive histone marks on target genes Ink4a/Arf and Pax3 • Derepressed Pax3 after Bmi1 ablation promotes adipocytic differentiation, Nakada and colleagues show that Bmi1 is highly expressed in bone marrow stromal cells, loss of which caused marrow adipocyte expansion and hematopoietic stem cell depletion. Bmi1 deletion diminished repressive histone marks on Pax3, leading to its expression and adipogenesis. Reduced Bmi1 expression with age may contribute to age-associated increase of marrow adipocytes.

Oncogenic mutations confer on cells the ability to propagate indefinitely, but whether oncogenes alter the cell fate of these cells is unknown. Here, we show that the transcriptional regulator PRDM16s causes oncogenic fate conversion by transforming cells fated to form platelets and erythrocytes into myeloid leukemia stem cells (LSCs). Prdm16s expression in megakaryocyte-erythroid progenitors (MEPs), which normally lack the potential to generate granulomonocytic cells, caused AML by converting MEPs into LSCs. Prdm16s blocked megakaryocytic/erythroid potential by interacting with super enhancers and activating myeloid master regulators, including PU.1. A CRISPR dropout screen confirmed that PU.1 is required for Prdm16s-induced leukemia. Ablating PU.1 attenuated leukemogenesis and reinstated the megakaryocytic/erythroid potential of leukemic MEPs in mouse models and human AML with PRDM16 rearrangement. Thus, oncogenic PRDM16s expression gives MEPs an LSC fate by activating myeloid gene regulatory networks.

Cryptomeridiol, a typical eudesmane diol, is the active principle component of the antispasmodic Proximol. Although it has been used for many years, the biosynthesis pathway of cryptomeridiol has remained blur. Among terpenoid natural products, terpenoid cyclases are responsible for cyclization and generation of hydrocarbon backbones. The cyclization is mediated by carbocationic cascades and ultimately terminated via deprotonation or nucleophilic capture. Isoprene precursors are, respectively, converted into hydrocarbons or hydroxylated backbones. A sesquiterpene cyclase in Tripterygium wilfordii (TwCS) was determined to directly catalyze (E,E)-farnesyl pyrophosphate (FPP) to unexpected eudesmane diols, primarily cryptomeridiol. The function of TwCS was characterized by a modular pathway engineering system in Saccharomyces cerevisiae. The major product determined by NMR spectroscopy turned out to be cryptomeridiol. This unprecedented production was further investigated in vitro, which verified that TwCS can directly produce eudesmane diols from FPP. Some key residues for TwCS catalysis were screened depending on the molecular model of TwCS and mutagenesis studies. As cryptomeridiol showed a small amount of volatile and medicinal properties, the biosynthesis of cryptomeridiol was reconstructed in S. cerevisiae. Optimized assays including modular pathway engineering and the CRISPR–cas9 system were successfully used to improve the yield of cryptomeridiol in the S. cerevisiae. The best engineered strain TE9 (BY4741 erg9::Δ-200-176 rox1::mut/pYX212-IDI + TwCS/p424-tHMG1) ultimately produced 19.73 mg/l cryptomeridiol in a shake flask culture.

PDK1 (phosphoinositide dependent kinase-1) plays an important regulatory role in B cells, T cells and platelets. Less is known about how PDK1 acts in hematopoietic stem cells (HSCs), especially in the fetal liver (FL) during embryonic hematopoiesis, as the FL is the primary fetal hematopoietic organ and the main site of HSC expansion and differentiation. Here, we deleted the PDK1 gene in hematopoietic cells by crossing Vav-Cre transgenic mice with PDK1f/f mice. Using a transplantation assay, we found that HSCs from the E15.5 FL of Vav-Cre;PDK1f/f embryos are severely impaired compared when compared with HSCs from PDK1f/f or PDK1f/+ FLs. Additionally, we found that there were more FL HSCs in an apoptotic state and active cell cycle in PDK1-deficient embryos than in control embryos. By comparing the expression profiles of FL-derived LSKs in Vav-Cre;PDK1f/f embryos to the controls, we found that the BH3-only protein PUMA and the cyclin family proteins were expressed higher in the Vav-Cre;PDK1f/f group, which may account for the increased apoptosis and activated cell cycle in the deficient HSCs. Furthermore, we demonstrated that the expression of FoxO3a was higher in PDK1-deficient LSKs, indicating that the Akt-FoxO3a-PUMA axis may participate in regulating LSKs apoptosis in the E15.5 FL. In contrast, FoxO1 expression was lower in PDK1-deficient LSK cells, suggesting that Akt-FoxO1-CCND may regulate the HSC cell cycle. Taken together, our findings support a critical role for PDK1 in maintaining FL hematopoiesis via regulating apoptosis and cell cycle of definitive hematopoiesis by the Akt-FOXO signaling pathways. Keywords: PDK1, Akt, HSC, FOXO, Fetal liver, Embryonic hematopoiesis

Characterization of hematopoietic stem cells (HSCs) has advanced largely owing to transplantation assays, in which the developmental potential of HSCs is assessed generally in nonhomeostatic conditions. These studies established that adult HSCs extensively contribute to multilineage hematopoietic regeneration upon transplantation. On the contrary, recent studies performing lineage tracing of HSCs under homeostatic conditions have shown that adult HSCs may contribute far less to steady-state hematopoiesis than would be anticipated based on transplantation assays. Here, we used 2 independent HSC-lineage-tracing models to examine the contribution of adult HSCs to steady-state hematopoiesis. We show that adult HSCs contribute robustly to steady-state hematopoiesis, exhibiting faster efflux toward the myeloid lineages compared with lymphoid lineages. Platelets were robustly labeled by HSCs, reaching the same level of labeling as HSCs by 1 year of chase. Our results support the view that adult HSCs contribute to the continuous influx of blood cells during steady-state hematopoiesis.

To examine the putative regulatory role of TwDXR in terpenoid biosynthesis and terpenoid biosynthetic pathway-related gene expression, through overexpression and RNA interference with TwDXR. We obtained 1410 and 454 bp TwDXR-specific fragments to construct overexpression and RNAi vectors. qRT-PCR was used to detect the expression of TwDXR and terpenoid biosynthesis pathway-related genes. The overexpression of TwDXR led to a 285% upregulation and the TwDXR RNAi led to a reduction to 26% of the control (empty vector-transformed cells) levels. However, pathway-related genes displayed different trends. When TwDXR was overexpressed, TwDXS expression decreased by 31% but increased to 198% when TwDXR expression was inhibited. The accumulation of terpenoids was also assayed. In the overexpression group, differences were not significant whereas the contents of triptolide and celastrol in the TwDXR RNAi samples were diminished by 27.3 and 24.0%, respectively. The feedback regulation of gene transcription and the accumulation of terpenoids in terpenoid biosynthesis in Tripterygium wilfordii were verified by TwDXR overexpression and RNAi experiments.

Summary Tripterygium wilfordii, which has long been used as a medicinal plant, exhibits impressive and effective anti-inflammatory, immunosuppressive and anti-tumor activities. The main active ingredients are diterpenoids and triterpenoids, such as triptolide and celastrol, respectively. A major challenge to harnessing these natural products is that they are found in very low amounts in planta. Access has been further limited by the lack of knowledge regarding their underlying biosynthetic pathways, particularly for the abeo-abietane tri-epoxide lactone triptolide. Here suspension cell cultures of T. wilfordii were found to produce triptolide in an inducible fashion, with feeding studies indicating that miltiradiene is the relevant abietane olefin precursor. Subsequently, transcriptome data were used to identify eight putative (di)terpene synthases that were then characterized for their potential involvement in triptolide biosynthesis. This included not only biochemical studies which revealed the expected presence of class II diterpene cyclases that produce the intermediate copalyl diphosphate (CPP), along with the more surprising finding of an atypical class I (di)terpene synthase that acts on CPP to produce the abietane olefin miltiradiene, but also their subcellular localization and, critically, genetic analysis. In particular, RNA interference targeting either both of the CPP synthases, TwTPS7v2 and TwTPS9v2, or the subsequently acting miltiradiene synthase, TwTPS27v2, led to decreased production of triptolide. Importantly, these results then both confirm that miltiradiene is the relevant precursor and the relevance of the identified diterpene synthases, enabling future studies of the biosynthesis of this important bioactive natural product.

1-Deoxy-d-xylulose-5-phosphate synthase (DXS) is the first enzyme in the plant 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway of terpenoid synthesis. TwDXS is a prominent protein in the Tripterygium wilfordii proteome, with especially high expression in the root periderm. It is significantly regulated by methyl jasmonate. Here, we studied the influence of TwDXS expression on bioactive terpenoids in T. wilfordii. Specific fragments of TwDXS (GenBank: AKP20998.1) with lengths of 2148 and 437 bp were amplified to construct the overexpression (OE) and RNA-interference (RNAi) vectors, respectively. After transformation of suspension cells, the expression of TwDXS and genes related to the terpenoid biosynthetic pathway was measured using qRT-PCR. TwDXS mRNA level was 153 and 43% of the control in the OE and RNAi lines. Related genes in the 2-C-methyl-d-erythritol 4-phosphate (MEP), mevalonic acid (MVA) and downstream pathways showed similar trends to the changes of TwDXS expression. Ultra Performance Liquid Chromatography (UPLC) was employed to measure the accumulation of terpenoids. Importantly, the triptolide content showed significant differences in both the TwDXS OE (222.35% of the control) and RNAi (34.86% of the control). However, there were no obvious changes in the celastrol content. In this study, we verified that the expression of TwDXS affects triptolide but not celastrol in T. wilfordii via both TwDXS OE and RNAi experiments.

Altered metabolism fuels 2 hallmark properties of cancer cells: unlimited proliferation and differentiation blockade. Adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of bioenergetics crucial for glucose metabolism in acute myeloid leukemia (AML), and its inhibition delays leukemogenesis, but whether the metabolic function of AMPK alters the AML epigenome remains unknown. Here, we demonstrate that AMPK maintains the epigenome of MLL-rearranged AML by linking acetyl-coenzyme A (CoA) homeostasis to Bromodomain and Extra-Terminal domain (BET) protein recruitment to chromatin. AMPK deletion reduced acetyl-CoA and histone acetylation, displacing BET proteins from chromatin in leukemia-initiating cells. In both mouse and patient-derived xenograft AML models, treating with AMPK and BET inhibitors synergistically suppressed AML. Our results provide a therapeutic rationale to target AMPK and BET for AML therapy.

Panax notoginseng, a perennial herb of the genus Panax in the family Araliaceae, has played an important role in clinical treatment in China for thousands of years because of its extensive pharmacological effects. Here, we report a high-quality reference genome of P. notoginseng, with a genome size up to 2.66 Gb and a contig N50 of 1.12 Mb, produced with third-generation PacBio sequencing technology. This is the first chromosome-level genome assembly for the genus Panax. Through genome evolution analysis, we explored phylogenetic and whole-genome duplication events and examined their impact on saponin biosynthesis. We performed a detailed transcriptional analysis of P. notoginseng and explored gene-level mechanisms that regulate the formation of characteristic tubercles. Next, we studied the biosynthesis and regulation of saponins at temporal and spatial levels. We combined multi-omics data to identify genes that encode key enzymes in the P. notoginseng terpenoid biosynthetic pathway. Finally, we identified five glycosyltransferase genes whose products catalyzed the formation of different ginsenosides in P. notoginseng. The genetic information obtained in this study provides a resource for further exploration of the growth characteristics, cultivation, breeding, and saponin biosynthesis of P. notoginseng.

For the monitoring of vascular growth as well as adaptive or therapeutic (re)vascularization endothelial-specific reporter mouse models are valuable tools. However, currently available mouse models have limitations, because not all endothelial cells express the reporter in all developmental stages. We have generated PECAM/eGFP embryonic stem (ES) cell and mouse lines where the reporter gene labels PECAM+ endothelial cells and vessels with high specificity. Native eGFP expression and PECAM staining were highly co-localized in vessels of various organs at embryonic stages E9.5, E15.5 and in adult mice. Expression was found in large and small arteries, capillaries and in veins but not in lymphatic vessels. Also in the bone marrow arteries and sinusoidal vessel were labeled, moreover, we could detect eGFP in some CD45+ hematopoietic cells. We also demonstrate that this labeling is very useful to monitor sprouting in an aortic ring assay as well as vascular remodeling in a murine injury model of myocardial infarction. Thus, PECAM/eGFP transgenic ES cells and mice greatly facilitate the monitoring and quantification of endothelial cells ex vivo and in vivo during development and injury.

The aim of this study was to verify the effects of TwIDI (GenBank: KT279355.1) on triptolide and celastrol accumulation in the biosynthesis of terpenoids in Tripterygium wilfordii and the regulation of the expression of related genes in the triptolide and celastrol biosynthesis pathway. After bioinformatics analysis of TwIDI, we cloned the full-length CDS and a specific 398 bp fragment to construct overexpression and RNAi vectors, respectively. The specific amplification of hygromycin and kanamycin resistance gene fragments confirmed that the expression vectors had been successfully delivered into Tripterygium wilfordii suspension cells. qRT-PCR was used to detect the expression of TwIDI and related genes in the triptolide and celastrol biosynthesis pathway. The expression of TwIDI was increased to 157% of the control group (empty vector) in the overexpression group, and was reduced to 71% of the control group in the RNAi group. Notably, the expression of other genes in the triptolide and celastrol biosynthesis pathway also showed differences. For example, TwMCS was reduced to 62% of the control when TwIDI was overexpressed and increased to 188% in the RNAi group. The expression of TwDXS did not change significantly both during TwIDI overexpression and RNAi group. The accumulation of triptolide and celastrol in the suspension cells of Tripterygium wilfordii was detected by UPLC, revealing that the contents of triptolide and celastrol were increased 1.36- and 1.20-fold over the control group in the overexpression group, and decreased to 0.16 and 0.36 of the control group in the RNAi group. Based on these findings, the effect on the accumulation of active terpenoids in Tripterygium wilfordii and the feedback regulation of genes in the triptolide and celastrol biosynthesis pathway was verified through TwIDI overexpression and RNAi experiments.

Activation of the unfolded protein response (UPR) sustains protein homeostasis (proteostasis) and plays a fundamental role in tissue maintenance and longevity of organisms. Long-range control of UPR activation has been demonstrated in invertebrates, but such mechanisms in mammals remain elusive. Here, we show that the female sex hormone estrogen regulates the UPR in hematopoietic stem cells (HSCs). Estrogen treatment increases the capacity of HSCs to regenerate the hematopoietic system upon transplantation and accelerates regeneration after irradiation. We found that estrogen signals through estrogen receptor α (ERα) expressed in hematopoietic cells to activate the protective Ire1α-Xbp1 branch of the UPR. Further, ERα-mediated activation of the Ire1α-Xbp1 pathway confers HSCs with resistance against proteotoxic stress and promotes regeneration. Our findings reveal a systemic mechanism through which HSC function is augmented for hematopoietic regeneration.

3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a pivotal regulator in the phosphoinositide 3-kinase (PI3K)-Akt signaling pathway that have been shown to play key roles in the functional development of B and T cells via activation of AGC protein kinases during hematopoiesis. However, the role of PDK1 in HSCs has not been fully defined. Here we specifically deleted the PDK1 gene in the hematopoietic system and found that PDK1-deficient HSCs exhibited impaired function and defective lineage commitment abilities. Lack of PDK1 caused HSCs to be less quiescent and to produce a higher number of phenotypic HSCs and fewer progenitors. PDK1-deficient HSCs were also unable to reconstitute the hematopoietic system. Notably, HSC function was more dependent on PDK1 than on mTORC2, which indicates that PDK1 plays a dominant role in the Akt-mediated regulation of HSC function. PDK1-deficient HSCs also exhibited reduced ROS levels, and treatment of PDK1-deficient HSCs with L-butathioninesulfoximine in vitro elevated the low ROS level and promoted colony formation. Therefore, PDK1 appears to contribute to HSC function partially via regulating ROS levels.

The adult hematopoietic system is sustained by a balance of self-renewal and differentiation in a small pool of stem and progenitor cells. This balance must be maintained to ensure a continuous supply of blood cells throughout life and prevent malignancy from arising. There are many facets of epigenetic regulation that are well known to be key components of healthy and diseased hematopoiesis, such as DNA methylation and histone post-translational modifications. However, the role of histone variant incorporation in hematopoiesis remains relatively unexplored. In this study, we explore the role of histone variant H3.3 regulation in the hematopoietic system by assessing the function of the histone H3.3 chaperone, Hira. Toward this goal, we use inducible and early developmental conditional knockout (cKO) mouse models to assess the role of Hira within the hematopoietic system. Following Hira cKO early in hematopoietic development (Vav-iCre; Hirafl/fl), we find that HSPCs are unaffected in the fetal liver but deplete quickly after homing to the bone marrow. Using polyinosinic-polycytidylic (pIpC) inducible Hira cKO mice (Mx1-Cre; Hirafl/fl), we find a similarly severe depletion of HSPCs in adult mice within 1 month after Hira loss. In contrast, differentiated cells remain largely unaffected following Hira cKO, demonstrating that Hira is especially important in the hematopoietic stem and progenitor compartment. Since Hira is known to incorporate H3.3 throughout the cell cycle and not just during S-phase like H3.1/2, we hypothesized that adult HSPCs are more dependent upon Hira to regulate histone H3 dynamics since they are slowly dividing. The loss of Hira-mediated H3.3 deposition would also be particularly detrimental to the function of these cells given its association with actively transcribed and bivalent genes. To test the role of Hira in maintaining gene expression patterns, we performed bulk RNA-seq on adult HSPCs and found that hematopoietic differentiation genes are dysregulated after Hira cKO with increased erythroid lineage and decreased lymphoid lineage gene expression. We then assessed gene expression changes in Hira cKO HSPCs in a doxycycline-inducible H2B-GFP background (Mx1-Cre; Hirafl/fl; R26-M2rtTa; TetOP-H2B-GFP) to distinguish between the gene expression changes caused by Hira loss before and after cell division. In the absence of Hira-mediated H3.3 incorporation, we expect some highly expressed genes in slowly dividing adult HSPCs to be affected by Hira loss prior to cell division due to nucleosome turnover in the wake of RNA Polymerase II. At other loci, like bivalent promoters, H3.3 would be diluted after cell division by H3.1/2 during S-phase in Hira cKO HSPCs. In support of this hypothesis, we found that increased expression of the erythroid differentiation gene Klf1 in Hira cKO MPPs after cell division (H2B-GFPLow) relative to Hira cKO MPPs before division (H2B-GFPHigh) and WT MPPs that have divided (H2B-GFPLow). The findings from both of these transcriptome analyses point toward a role of Hira in regulating HSPC differentiation genes and are supported by our in vitro and in vivo data showing increased differentiation of Hira cKO HSPCs and decreased self-renewal. In order to more fully understand the H3.3-dependent gene expression changes after Hira cKO in HSPCs, we correlated H3.3 enrichment patterns from chromatin-immunoprecipitation and sequencing (ChIP-seq) with our data from assay for transposase-accessible chromatin and sequencing (ATAC-seq). Our results demonstrate that Hira cKO HSPCs have more open chromatin and fewer H3.3 peaks, suggesting that loss of Hira-mediated H3.3 deposition increases DNA accessibility. This study identifies a novel epigenetic mechanism required for adult HSPC maintenance and elucidates a previously unappreciated regulator of normal hematopoietic homeostasis. Further understanding how Hira-mediated H3.3 regulation maintains adult HSPCs will provide greater depth to our current understanding of the epigenetic regulators essential for hematopoiesis. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is primarily a disease of older adults with poor treatment outcomes. Despite years of intensive research, the standard induction therapy for AML has remained largely unchanged for decades. Thus, the development of new and efficacious therapeutic targets for AML is urgently needed. Leukemia cells exhibit multiple metabolic aberrations that may be therapeutically targeted. Here, we show that nicotinamide adenine dinucleotide (NAD+) promotes leukemogenesis and causes chemotherapy treatment resistance through fueling energetic metabolism, and pinpoints nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) is a novel therapeutic target for AML. To identify novel genes essential for AML, we performed a whole genome CRISPR dropout screen by using MOLM13 cell line and identified 1,951 essential genes (Fig. A). By searching druggable targets among these genes, we narrowed down to 345 genes, among which we found two genes, NMNAT1 (nicotinamide nucleotide adenylyltransferase 1) and NAMPT (nicotinamide phosphoribosyltransferase), both involved in key steps in NAD+ biosynthesis. We comprehensively analyzed dependency scores for all genes involved in the NAD+ biosynthetic pathways (de novo synthesis pathway, the Preiss-Handler pathway and the salvage pathway) across a broad panel of cancer cell lines from the Dependency Map database (https://depmap.org/portal/). The results showed that NMNAT1 and NAMPT are both strongly selective and uniquely required for hematological malignancies compared to other cancers (Fig. B). Since little success has been achieved for NAMPT inhibitors in clinical trials, our attention was drawn to NMNAT1, which encodes a nuclear localized enzyme catalyzing the final step in NAD+ biosynthesis. We confirmed that deletion of NMNAT1 in AML cells significantly reduced nuclear NAD+ level and cell viability over time while sparing normal hematopoietic progenitor cells, suggesting that NMNAT1 is targetable to AML. Overexpression of wild-type Nmnat1 but not the enzymatically inactive forms rescued NMNAT1-KO AML, indicating that the catalytic activity of NMNAT1 is required for AML. To study the role of NAD+ in AML, we first measured NAD+ levels in leukemic and normal cells, and found higher NAD+ levels in leukemia-initiating cells from a murine MLL-AF9-induced AML model compared to normal cells. Supplementation of NAD+ metabolites (NMN, NAM and NR) increased AML proliferation, enhanced glycolysis (lactate production) and oxidative phosphorylation (ATP production), resulting in chemotherapy resistance (Fig. C). Deletion of NMNAT1 sensitized AML cell to chemotherapy treatment. To study the role of NMNAT1 in leukemogenesis in vivo, we genetically deleted NMNAT1 in murine or human leukemia cells, transplanted them into recipient mice, and found that deletion of NMNAT1 reduced leukemic burden and extended leukemia-free survival (Fig. D). Finally, to reveal the molecular mechanisms underlying NMNAT1 KO-mediated cell death (increased levels of gamma-H2AX), RNA-seq and functional assay of NAD+ dependent enzymes were performed. We found that the reduction of nuclear NAD+ resulting from NMNAT1 deletion upregulated genes involved in DNA repair pathway, which may be linked to impaired PARPs and Sirtuins activity. Our findings reveal the important function of NAD+ in leukemogenesis and chemoresistance, and identify NMANT1 as a novel therapeutic target for AML. Figure Disclosures No relevant conflicts of interest to declare.

The maintenance of the hematopoietic system by hematopoietic stem cells (HSCs) is an important topic in both clinical and basic hematology study due to their enormous therapeutic potential. The hematopoietic hierarchy has recently garnered renewed interest following the development of single-cell assays and improved strategies for genetic labeling, resulting in new hierarchical models that challenge the classical view of hematopoiesis. However, the kinetic of hematopoiesis under steady-state and stress condition and the contribution of HSCs toward steady-state hematopoiesis remain controversial and unclear. Using two tamoxifen-induced lineage tracing models, namely Fgd5-CreERT2/ROSA26-tdTomato and Krt18-CreERT2/ROSA26-tdTomato, we traced the contribution of HSCs to downstream blood cells. The initial labeling will occurred mostly within HSCs due to the largely restricted expression of Fgd5 and Krt18 in HSCs. Our preliminary data suggest that adult HSCs contribute robustly to steady-state hematopoiesis, exhibiting faster efflux toward platelets and myeloid lineages compared with lymphoid lineages. Interestingly, we found that myeloid cells and platelets were labeled rapidly after the initial HSC labeling without the appearance of the previously identified intermediate populations, suggesting the potential bypassing differentiation from HSCs to these lineages. In addition to steady-state hematopoiesis, we also investigated the response of the hematopoietic system to stress. Using the lineage tracing models mentioned above, we observed that HSCs are able to respond to stress to compensate the loss of a specific cell type, without affecting their contribution toward other lineages. For example, antibody-mediated platelet depletion resulted in a compensatory output from HSCs toward platelets. On the other hand, PHZ-induced hemolytic anemia led to an accelerated production of red blood cells. In both cases, other blood lineages remained unaffected compared to controls. In order to acquire a more comprehensive view of steady-state hematopoiesis, we combined Fgd5-CreERT2/ROSA26-tdTomato lineage tracing model with single-cell RNA-seq. To be inclusive of known and unknown HSPC populations, we sorted Lin-tdTomato+ cells at 1 week, 2 weeks and 1 month after the first tamoxifen treatment. HSCs, MPPs and HPC1/2 sorted from wild-type animals served as the reference groups. We observed the appearance of myeloid and megakaryocytic progeny as early as 1 week after the initial HSC labeling, suggesting the rapid contribution of HSCs toward these lineages. Erythroid-biased population emerged at the 2-week time point, with the expansion of myeloid progeny. Intriguingly, pseudo-time analysis revealed a relatively upstream position of erythroid-biased cluster, indicating the early emergence of erythroid progeny. The clear appearance of lymphoid progeny was absent until 1 month after the initial labeling, corresponding to the later onset of lymphoid differentiation. In summary, our results support the active role of HSCs in steady-state hematopoiesis and rapid output toward megakaryocytic, erythroid, and myeloid lineages from HSCs. Moreover, our data also provide insight into the responses of HSCs toward different stimuli, which can potentially contribute to our understanding of hematopoietic-related diseases and uncover novel treatment avenues. Disclosures No relevant conflicts of interest to declare.

Coronary arteries bring blood flow to the heart muscle. Understanding the developmental program of the coronary arteries provides insights into the treatment of coronary artery diseases. Multiple sources have been described as contributing to coronary arteries including the proepicardium, sinus venosus (SV), and endocardium. However, the developmental origins of coronary vessels are still under intense study. We have produced a new genetic tool for studying coronary development, an AplnCreER mouse line, which expresses an inducible Cre recombinase specifically in developing coronary vessels. Quantitative analysis of coronary development and timed induction of AplnCreER fate tracing showed that the progenies of subepicardial endothelial cells (ECs) both invade the compact myocardium to form coronary arteries and remain on the surface to produce veins. We found that these subepicardial ECs are the major sources of intramyocardial coronary vessels in the developing heart. In vitro explant assays indicate that the majority of these subepicardial ECs arise from endocardium of the SV and atrium, but not from ventricular endocardium. Clonal analysis of Apln-positive cells indicates that a single subepicardial EC contributes equally to both coronary arteries and veins. Collectively, these data suggested that subepicardial ECs are the major source of intramyocardial coronary arteries in the ventricle wall, and that coronary arteries and veins have a common origin in the developing heart.

Aberrant transcriptional regulation contributes to the pathogenesis of both congenital and adult forms of liver disease. Although the transcription factor RBPJ is essential for liver morphogenesis and biliary development, its specific function in the differentiation of hepatic progenitor cells (HPC) has not been investigated, and little is known about its role in adult liver regeneration. HPCs are bipotent liver stem cells that can self-replicate and differentiate into hepatocytes or cholangiocytes in vitro. HPCs are thought to play an important role in liver regeneration and repair responses. While the coordinated repopulation of both hepatocyte and cholangiocyte compartment is pivotal to the structure and function of the liver after regeneration, the mechanisms coordinating biliary regeneration remain vastly understudied. Here, we utilized complex genetic manipulations to drive liver-specific deletion of the Rbpj gene in conjunction with lineage tracing techniques to delineate the precise functions of RBPJ during biliary development and HPC-associated biliary regeneration after hepatectomy. Furthermore, we demonstrate that RBPJ promotes HPC differentiation toward cholangiocytes in vitro and blocks hepatocyte differentiation through mechanisms involving Hippo-Notch crosstalk. Overall, this study demonstrates that the Notch-RBPJ signaling axis critically regulates biliary regeneration by coordinating the fate decision of HPC and clarifies the molecular mechanisms involved.

Although great efforts have been made to improve available therapies, the mortality rate of acute myeloid leukemia (AML) remains high due to poor treatment response and frequent relapse after chemotherapy. Leukemia stem cells (LSCs) are thought to account for this poor prognosis and relapse. Phosphoinositide-dependent kinase 1 (PDK1) is a critical regulator of the PI3K/Akt pathway and has been shown to be frequently activated in leukemia. However, the role of PDK1 in the regulation of LSCs in AML is still not clear. Using a PDK1 conditional deletion MLL-AF9 murine AML model, we revealed that the deletion of PDK1 prolonged the survival of AML mice by inducing LSC apoptosis. This was accompanied by the increased expression of the pro-apoptotic genes Bax and p53 and the reduced expression of Stat5, which has been shown to be constitutively activated in leukemia. Thus, our findings suggest that PDK1 plays an essential role in maintaining LSCs. Further delineating the function of PDK1 in LSCs may provide a new strategy for the improved treatment of AML relapse.

Under pathophysiological conditions in adults, endothelial cells (ECs) sprout from pre-existing blood vessels to form new ones by a process termed angiogenesis. During embryonic development, Apelin (APLN) is robustly expressed in vascular ECs. In adult mice, however, APLN expression in the vasculature is significantly reduced. Here we show that APLN expression is reactivated in adult ECs after ischaemia insults. In models of both injury ischaemia and tumor angiogenesis, we find that Apln-CreER genetically labels sprouting but not quiescent vasculature. By leveraging this specific activity, we demonstrate that abolishment of the VEGF–VEGFR2 signalling pathway as well as ablation of sprouting ECs diminished tumour vascularization and growth without compromising vascular homeostasis in other organs. Collectively, we show that Apln-CreER distinguishes sprouting vessels from stabilized vessels in multiple pathological settings. The Apln-CreER line described here will greatly aid future mechanistic studies in both vascular developmental biology and adult vascular diseases., Apelin expression is robust in embryonic but not in adult endothelial cells (ECs), where it can be reactivated by hypoxia. Liu et al. show that apelin-driven expression of Cre recombinase in mice can be used for labelling of, or gene ablation in, sprouting but not quiescent ECs in pathologies characterized by hypoxia.

The development of early B cells, which are generated from hematopoietic stem cells (HSCs) in a series of well-characterized stages in bone marrow (BM), represents a paradigm for terminal differentiation processes. Akt is primarily regulated by phosphorylation at Thr308 by PDK1 and at Ser473 by mTORC2, and Akt signaling plays a key role in hematopoiesis. However, the role of mTORC2 in the development of early B cells remains poorly understood. In this study, we investigated the functional role of mTORC2 by specifically deleting an integral component, Rictor, in a hematopoietic system. We demonstrated that the deletion of Rictor induced an aberrant increase in the FoxO1 and Rag-1 proteins in BM B cells and that this increase was accompanied by a significant decrease in the abundance of B cells in the peripheral blood (PB) and the spleen, suggesting impaired development of early B cells in adult mouse BM. A BM transplantation assay revealed that the B cell differentiation defect induced by Rictor deletion was not affected by the BM microenvironment, thus indicating a cell-intrinsic mechanism. Furthermore, the knockdown of FoxO1 in Rictor-deleted HSCs and hematopoietic progenitor cells (HPCs) promoted the maturation of B cells in the BM of recipient mice. In addition, we revealed that treatment with rapamycin (an mTORC1 inhibitor) aggravated the deficiency in B cell development in the PB and BM. Taken together, our results provide further evidence that Rictor regulates the development of early B cells in a cell-intrinsic manner by modifying the expression of FoxO1 and Rag-1.

Cardiac malformations due to aberrant development of the atrioventricular (AV) valves are among the most common forms of congenital heart diseases. Normally, heart valve mesenchyme is formed from an endothelial to mesenchymal transition (EMT) of endothelial cells of the endocardial cushions. Yes-associated protein 1 (YAP1) has been reported to regulate EMT in vitro, in addition to its known role as a major regulator of organ size and cell proliferation in vertebrates, leading us to hypothesize that YAP1 is required for heart valve development. We tested this hypothesis by conditional inactivation of YAP1 in endothelial cells and their derivatives. This resulted in markedly hypocellular endocardial cushions due to impaired formation of heart valve mesenchyme by EMT and to reduced endocardial cell proliferation. In endothelial cells, TGFβ induces nuclear localization of Smad2/3/4 complex, which activates expression of Snail, Twist1, and Slug, key transcription factors required for EMT. YAP1 interacts with this complex, and loss of YAP1 disrupts TGFβ-induced up-regulation of Snail, Twist1, and Slug. Together, our results identify a role of YAP1 in regulating EMT through modulation of TGFβ-Smad signaling and through proliferative activity during cardiac cushion development.

ATP-dependent SWI/SNF chromatin remodeling complexes utilize ATP hydrolysis to non-covalently change nucleosome-DNA interactions and are essential in stem cell development, organogenesis, and tumorigenesis. Biochemical studies show that SWI/SNF in mammalian cells can be divided into two subcomplexes BAF and PBAF based on the subunit composition. ARID2 or BAF200 has been defined as an intrinsic subunit of PBAF complex. However, the function of BAF200 in vivo is not clear. To dissect the possible role of BAF200 in regulating embryogenesis and organ development, we generated BAF200 mutant mice and found they were embryonic lethal. BAF200 mutant embryos exhibited multiple cardiac defects including thin myocardium, ventricular septum defect, common atrioventricular valve, and double outlet right ventricle around E14.5. Moreover, we also detected reduced intramyocardial coronary arteries in BAF200 mutants, suggesting that BAF200 is required for proper migration and differentiation of subepicardial venous cells into arterial endothelial cells. Our work revealed that PBAF complex plays a critical role in heart morphogenesis and coronary artery angiogenesis.

Maintaining metabolic homeostasis is a fundamental requirement for cells to survive. One critical requirement is to accomplish a balance between anabolism and catabolism. AMPK regulates this balance by directly sensing AMP-to-ATP ratio and its activation during cell energy deficit promotes ATP production and inhibits ATP usage. Several pieces of evidence point AMPK as a tumor suppressor: the upstream protein, LKB1, is a well-established tumor suppressor; AMPK negatively interacts with the tumor promoting mTOR pathway; anti-diabetic drugs such as metformin with tumor preventive potential are shown to activate AMPK. However, emerging evidence accumulates to support an opposite role of AMPK in promoting tumor growth. AMPK is found to protect tumor cells from metabolic crisis through different mechanisms: autophagy induction; maintaining proper ATP levels and redox environment; histone H2B tail phosphorylation. These two contrasting findings suggested multiple facets of AMPK, thus pointing to the urge to understand mechanisms of AMPK in specific contexts. We examined the role of AMPK by deleting both α1 and α2 subunit in a mouse model of acute myeloid leukemia with t(9;11) translocation. AMPK deficiency depletes the leukemia-initiating-cell population and decreases the leukemogenic potential of these cells. In order to study the metabolic regulatory effects of AMPK, we profiled the metabolites and found AMPK deficiency associates with a decreased level of glycolytic activity and reduction of acetyl-CoA, which is the major donor for histone acetylation. Therefore, we hypothesized that AMPK can affect histone acetylation through regulating the level of acetyl-CoA, and functionally alter leukemogenic potential. We first profiled histone acetylation using western blot with antibodies targeting global histone acetylation and specific histone residues. Intriguingly, we found in AMPK-deficient cells, global histone H3 and H4 acetylation levels are decreased, as well as acetylation at specific histone residues such as H3K9, H3K27 and H4K8. To build a causal link between decreased acetyl-CoA and histone acetylation, we supplemented leukemia-initiating-cells in culture with acetyl-CoA precursors such as acetate and pyruvate. The supplementation successfully increased intracellular acetyl-CoA levels as well as histone acetylation levels. Functionally restoring the intracellular acetyl-CoA and histone acetylation increased the proliferative potential of AMPK-deficient leukemia-initiating-cells and maintained cell at a more undifferentiated state. These results suggest that AMPK regulates a set of leukemogenic genes by maintaining histone acetylation levels. We hypothesize that AMPK links metabolic status to epigenetic gene regulation to promote leukemogenesis. Disclosures No relevant conflicts of interest to declare.

Hematopoietic stem cells (HSCs) exist as a rare population with two essential properties of self-renewal and differentiation. HSCs can give rise to all hematopoietic progenitor and mature cells. While critical for a full understanding of the hematopoietic process and HSC-related clinical applications, the mechanisms of self-renewal and differentiation of HSCs remain elusive. The PI3K-Akt signaling pathway plays essential roles in the regulation of hematopoiesis. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) activates multiple AGC kinases including Akt and is a pivotal regulator in this pathway. PDK1 phosphorylates Akt at its T308 residue and regulates the functional development of B and T cells during hematopoiesis. However, the role of PDK1 in HSCs has not been fully defined. In this study, we generated PDK1 conditional knockout mice Vav-Cre;PDK1fl/fl (PDK1Δ/Δ) to explore the roles of PDK1 in HSCs. While PDK1Δ/Δ mice have reduced B and T cell counts as previously described, their LT-HSCs and ST-HSCs were significantly increased in comparison with WT mice while MPPs and CMPs were decreased after PDK1 deletion, indicating that the loss of PDK1 perturbed the steady-state hematopoiesis. Furthermore, although deletion of PDK1 increased the frequency of HSCs, PDK1-deficient HSCs fail to reconstitute the hematopoietic system when PDK1-deficient HSCs were used in bone marrow transplantation and competitive transplantation experiments in comparison to the WT HSCs, indicating that PDK1 is vital for hematopoiesis. To explore the mechanisms by which PDK1 regulates HSC function, we examined the cell cycle status and found the percentage of PDK1Δ/Δ HSCs was decreased significantly in G0 stage while increased in G1 and S/G2/M phases. This suggests an increase in HSC exit from a quiescent state. Since MPPs were significantly decreased in bone marrow, we examined the percentage of Annexin V+ DAPI- PDK1Δ/Δ and WT MPPs and found that they are comparable. This indicates that apoptosis did not cause the decrease in MPPs. In addition, a total of 300 LT-HSCs from PDK1Δ/Δ or WT mice and competitor cells were transplanted into lethally irradiated recipient mice to examine whether the decrease in MPPs is due to a defect in HSC differentiation. We found that less than 1% of MPPs arose from PDK1Δ/Δ HSCs 12 weeks after transplantation, indicating that PDK1 is required for the differentiation from LT-HSCs to MPPs. Because the full activation of Akt requires cooperative phosphorylation at its S473 and T308 residues by mTORC2 and PDK1, respectively, we also investigated the function of HSCs in RictorΔ/Δ PDK1Δ/Δ (DKO) mice in conjunction with RictorΔ/Δ or PDK1Δ/Δ mice to explore how mTORC2 and/or PDK1 influence Akt function in HSCs. The flow cytometric analyses of peripheral blood and bone marrow samples revealed very similar parameters of RictorΔ/Δ PDK1Δ/Δ and PDK1Δ/Δ mice. Interestingly, Rictor seemed to exert a minimal impact on HSCs and MPPs. More importantly, in contrast to RictorΔ/Δ, RictorΔ/Δ PDK1Δ/Δ HSCs failed to reconstitute the hematopoietic system after transplantation as PDK1Δ/Δ HSCs, suggesting that PDK1 plays a dominant role in the Akt-mediated regulation of HSC function. To explore the mechanism that leads to the defect in HSCs due to loss of PDK1, we assessed ROS levels in PDK1-deficient HSCs and found that PDK1-deficient LSKs and HSCs exhibit greatly reduced ROS levels when compared with the control HSCs. Treating PDK1-deficient BM cells with BSO in vitro increased cellular ROS levels and the colony counts of PDK1-deficient BM cells significantly. Notably, the recovery effect was only observed with BSO concentrations lower than 0.03 mM. This suggests that ROS levels are precisely controlled in HSCs. Higher or lower ROS levels beyond the normal range are both harmful to normal HSC functions. Since increased SDFα expression is associated with cellular ROS levels in various cells including hematopoietic cells, we also treated PDK1Δ/Δ mice with SDFα and found that it couldpartially rescue the defective differentiation ability of PDK1-deficient HSCs. In addition, we found that PDK1 deletion could significantly prolong the life span and inhibit the leukemia development in murine T-ALL model via altering leukemic cell differentiation and proliferation. Taken together, PDK1 controls HSC differentiation via regulating cellular ROS levels and regulates malignant hematopoiesis. Disclosures No relevant conflicts of interest to declare.

Our recent study identified a nonsense mutation of La-related protein 4B (LARP4B) from whole genome sequencing of a 3-year-old female monozygotic twin pair discordant for MLL-associated acute myeloid leukemia (AML). To study the role of LARP4B in AML, we established a LARP4B-knockdown MLL-AF9 AML mouse model. Using this mouse model, we found that LARP4B knockdown significantly decreased leukemia cells in the peripheral blood, spleen, and bone marrow and prolonged the survival of AML recipient mice. Additional studies showed that LARP4B knockdown reduced leukemia stem cells (LSCs) and impaired the self-renew capacity of LSCs. Cell cycle analysis revealed that LARP4B knockdown arrested more LSCs in the G0 phase. The transcription of the cell cycle inhibitors p16, p19, and p21 and of the lineage-specific transcription factor CCAAT-enhancer-binding protein α was increased in the LARP4B-knockdown LSCs. Thus, our results demonstrate that LARP4B plays an important role in the maintenance of LSCs and suggest that LARP4B may regulate the cell cycle of LSCs via suppressing the expression of the cell cycle inhibitors p16, p19, and p21 and the myeloid specific transcription factor CCAAT-enhancer-binding protein α.